"Immunotolerance" and "immunosuppression" are general terms to describe the compatibility of materials which would be normally expected to result in an immune response. When tissues or cells are transplanted into an allogeneic host, absent these or immunosuppressive conditions, the host immune system will mount an immune response to the foreign antigens (host-versus-graft disease), and, more seriously, the immunocompetent cells in the transplant may respond to the antigens contained in the host (graft-versus-host disease). Other unwanted immune responses include allergic reactions and autoimmune diseases. Under all of the foregoing conditions, it is desirable that the immune response be suppressed.
It has been known for years that neonatal mammals are capable of acquiring persistent immunotolerance with respect to allogeneic immunocompetent cells or to other antigenic substances administered within a few days after birth. For example, mice injected with allogeneic substances bearing the histocompatibility antigens are able later to accept skin grafts from donors of the previously injected genotype. More recently, it has been shown that this immunosuppressive property of neonates is simulated by adults subjected to total lymphoid irradiation (TLI), i.e., high dosages of radiation sustainable when nonlymphoid tissues are protected. Such radiation has been used in the treatment, for example, of Hodgkin's disease. Adult subjects who have been subjected to TLI are also capable of acquiring persistent immunotolerance to antigens administered within a few days of the completion of TLI.
The detailed mechanism by which neonates and TLI-treated subjects are capable of acquiring an immunotolerance with respect to antigens and cells administered within these windows is not understood. Both clonal deletion and active suppression paradigms have been proposed. The picture is further complicated by the presence of at least two types of suppressor cells in neonatal spleens. One type is represented by macrophage precursors, which suppress in vitro the antibody response to sheep red-blood cells. This activity is inhibited by indomethacin, and the cells are thus presumed to be prostaglandin dependent. The other type is represented by lymphocytes which inhibit the mixed leukocyte response (MLR). These cells are apparently prostaglandin independent. One group of cells in this class, "null" cells, lack the surface markers characteristic of T cells, B cells, or macrophages, and have morphologies similar to natural killer cells in that they are large granular lymphocytes which lack antigen specificity and which carry out their effective function without antigenic stimulation. The surface phenotype of these cells is Ig.sup.-, CD4.sup.-, CD8.sup.31 , CD3.sup.-, MAC-1-, TCR.alpha..beta..sup.-. This population of null suppressor cells has been designated, for purposes of symmetry with natural killer (NK) cells, natural suppressor (NS) cells (Oseroff, A., et al., J Immunol (1984) 132:101).
In addition to the null phenotypes, natural suppressor cells that contain the surface phenotype Ig.sup.-, CD4.sup.-, CD8.sup.-, CD3.sup.+, MAC-1.sup.-, TCR.alpha..beta..sup.+ have been recovered from the spleens of adult mice subjected to TLI. These "double negative" suppressors (DNS) can be propagated in vitro under proper conditions indefinitely (Hertel-Wulff, B., et al., J Immunol (1984) 133:2791-2796, incorporated herein by reference). These "DNS" cells can also be cloned from cells obtained from neonatal spleen, normal adult spleen and bone marrow. (The null cells described above have also been cloned from adult TLI spleen and from normal thymus.)
It has now been found that cloned DNS or null cells are capable, when administered in vivo, of suppressing graft-versus-host disease initiated by simultaneously administered immunocompetent cells. In addition, the propagated DNS and null cells secrete into supernatant media a soluble factor which is capable of suppressing the immune response as shown by the ability to suppress alloreactivity in the commonly used in vitro test (the mixed leukocyte reaction (MLR) mentioned above) and in vivo in suppressing the acute immune response referred to as graft-versus-host disease as well as encouraging engraftment of transplanted tissue.
Additional publications of the applicants subsequent to the filing of the parent application herein further describe the cells and secreted factor. The status of the suppressor activity of the NS cells in general was reviewed by Strober, S., Ann Rev Immunol (1984) 2:219; a further description of the cloned suppressor cell lines is provided by Schwadron, R. B., et al., J Exp Med (1985) 162:297, and by Schwadron, R. B., et al., Transplantation (1989), p. 107. Hertel-Wulff, B., et al., J Exp Med (1987) 16:1168, describe the rearrangement and expression of T-cell receptor genes in the cloned NS cell lines. The verification of the surface phenotype of the cloned DNS cell lines as IL-2R.sup.+, CD3.sup.+, CD4.sup.-, CD8.sup.- and TCR.alpha..beta..sup.+ was described by Strober, S., et al., J Immunol (1989) 143:1118. The DNS cells are thus distinguished from the null NS cells which expresses neither T nor B cell markers (CD3.sup.-, TCR.alpha..beta..sup.-, Ig.sup.-). However, both types (null and DNS) express the surface receptor for IL-2 (IL-2R). The null cells are thus CD3.sup.-, Ig.sup.-, TCR.alpha..beta..sup.-, MAC-1.sup.-, and IL-2R.sup.+. A further description of the soluble factor was provided by Hertel-Wulff, B., et al., J Immunol (1988) 140:2633. A more recent paper by Palathumpat, V. et al., J Immunol (1992) 148:373-380 further describes separations of the double negative suppressor cells from bone marrow of murine subjects.
The ability to suppress the capacity of immunocompetent donor cells to effect graft-versus-host disease is of particular significance in view of the growing technology permitting successful allografts and organ transplants. Transfers of healthy tissues into recipients in need of them seems at present limited in the main by lack of immunotolerance with respect to the recipient. Thus, if the problem of graft-versus-host disease could be solved, the dangers associated with bone marrow transplants would be considerably reduced. In the case of whole organ transplants, rejection of the organ would be reduced.
In addition, encouraging engraftment of transplanted cells is of importance. It is well known that administration of allogeneic tissue in the form of bone marrow to either neonates or adults subjected to TLI during the "window" period will convert the recipient to a chimera, which will recognize as "self" both its own antigens and those of the alloantigen administered at this time. The formation of a chimera shows that engraftment has occurred. The chimeric character of the host is also such that subsequently introduced immunocompetent cells will not attack host tissue. These chimeras are thus not only specifically receptive to the simultaneously administered donor bone marrow, but also to other donor tissue. The chimeric recipient, therefore, will in the future be able to tolerate transplanted tissue from the original donor.
In effecting engraftment, cells enriched in the cell surface marker CD34 are known to be helpful, since this marker appears to characterize "stem" or "progenitor" cells. These cells are progenitors of the multiplicity of differentiated cells that are found in the blood, including monocytes, macrophage, lymphocytes, red blood cells and so forth. Thus, the capacity of transplanted cells to engraft will be dependent on the enrichment of the transplanted cells in cells which bear the CD34 marker.
The recipient normal adult host must, of course, be prevented from succumbing to an acute immune response effected by the originally administered bone marrow cells. The DNS and null NS cells are capable of muting the immediate immune response and any other antidonor response sufficiently to permit the generation of characteristics of the chimera. Both fresh and cloned DNS and null cells are also capable of preventing the in vivo graft versus host immune response. The secreted factor is capable of inhibiting the immune response of donor against host, and host against donor cells in vitro, and in vivo.
Both null and DNS cells and the factors secreted by them are therefore useful in providing immediate blocking of either the graft immune response against mammalian hosts or host response to the donor by coadministration of the cells or factors along with foreign substances or tissues to which such immediate tolerance is desired. In addition, and in particular, these cells and/or factors permit the hosts to become tolerant of both present and future grafts where the donor tissue is derived from the same genotype donor as tissue coadministered with the cells or factor.